A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

Therapeutic potential of targeting IL-17.

A June 2012 meeting in Dublin, Ireland, showcased advances in the understanding of the role of IL-17 and related cytokines in host immunity and how these cytokines have been successfully targeted for the treatment of autoimmunity.

Comparison of mortality of brachycephalic dogs undergoing partial staphylectomy using conventional incisional, carbon dioxide laser, or bipolar vessel sealing device.

OBJECTIVE: To compare mortality of dogs undergoing partial staphylectomy using conventional incisional, carbon dioxide (CO2 ) laser, and bipolar vessel sealing device (BVSD) techniques for the treatment of brachycephalic obstructive airway syndrome (BOAS). STUDY DESIGN: Retrospective multicenter cohort study. ANIMALS: A total of 606 client-owned English bulldogs, French bulldogs, and pugs. METHODS: Medical records from 2011 to 2021 were reviewed for signalment, history, surgical technique, length of hospitalization, and complications. Multivariate statistical analysis was performed to compare odds of mortality between the three techniques of staphylectomy. RESULTS: The overall mortality rate was 24/606 (4.0%). Of those 24 dogs, staphylectomy was performed with BVSD technique in 13 cases, with CO2 laser in nine, and using conventional incisional technique in two. Nine dogs were graded II or III laryngeal collapse, 14 were graded I, and one was unknown. BVSD technique was associated with mortality prior to discharge compared to the other two techniques (OR = 6.0, 95% CI: 1.3-28.4, p = .023). No differences were detected between conventional incisional and CO2 laser techniques. Concurrent higher grade (stage II or III) laryngeal collapse was independently associated with mortality prior to discharge (OR = 4.6, 95% CI: 1.8-11.8, p = .002). CONCLUSION: The use of BVSD and grade of laryngeal collapse were associated with a higher risk of perioperative mortality. CLINICAL SIGNIFICANCE: Clinical studies using a randomized trial design should be conducted to further determine the putative influence of surgical instrumentation in the perioperative mortality rate following multilevel surgery in dogs with BOAS.

Effect of preoperative ondansetron on postoperative nausea in healthy dogs undergoing laparoscopic gastropexy and castration.

OBJECTIVE: To investigate if preoperative ondansetron reduces postoperative nausea associated with laparoscopic gastropexy and castration in dogs. STUDY DESIGN: Prospective clinical study. ANIMALS: Twenty client-owned, healthy male dogs. METHODS: Dogs were premedicated with dexmedetomidine (2-5 mcg kg-1) and methadone (0.2-0.5 mg kg-1) intramuscularly. General anesthesia was induced with propofol and maintained with an inhalant anesthetic agent. Dogs were randomized into group S (saline 0.1 mL kg-1, intravenously) or group O (ondansetron 0.2 mg kg-1, intravenously). Plasma and serum were collected before premedication and 3 hours postextubation to measure arginine vasopressin (AVP) and cortisol concentrations. Nausea scoring occurred before and 10 minutes after premedication, immediately after extubation, and at 1, 2 and 3 hours postextubation. Data were analyzed by mixed and split-plot anova with Bonferroni adjustment for the number of group comparisons. Significance was set at p < 0.05. RESULTS: Nausea scores increased over time at 1 (p = 0.01) and 2 (p < 0.001) hours postextubation in both groups compared with before premedication. Median nausea score (0-100 mm) for groups S and O before premedication were 2.5 and 0.5 mm, respectively. At 1 and 2 hours postextubation, group S scored 7.5 and 4.0 mm and group O scored 6.0 and 5.0 mm, respectively. No significant differences in nausea scores within or between groups were observed before premedication and 3 hours postextubation. Cortisol concentrations increased significantly 3 hours postextubation in both groups (p < 0.001) compared with before premedication, with no differences between groups. AVP concentrations showed no significant differences within or between groups. CONCLUSIONS AND CLINICAL RELEVANCE: Preoperative intravenous administration of ondansetron (0.2 mg kg-1) did not impact postoperative nausea after laparoscopic gastropexy and castration. Investigation of higher doses of ondansetron on the incidence of postoperative nausea and vomiting in dogs after surgery is warranted.

Investigating immunoregulatory effects of myeloid cell autophagy in acute and chronic inflammation.

Studies have highlighted a critical role for autophagy in the regulation of multiple cytokines. Autophagy inhibits the release of interleukin (IL)-1 family cytokines, including IL-1α, IL-1ß and IL-18, by myeloid cells. This, in turn, impacts the release of other cytokines by myeloid cells, as well as other cells of the immune system, including IL-22, IL-23, IL-17 and interferon-γ. Here, we assessed the impact of genetic depletion of the autophagy gene Atg7 in myeloid cells on acute and chronic inflammation. In a model of acute lipopolysaccharide-induced endotoxemia, loss of autophagy in myeloid cells resulted in increased release of proinflammatory cytokines, both locally and systemically. By contrast, loss of Atg7 in myeloid cells in the Lyn-/- model of lupus-like autoimmunity resulted in reduced systemic release of IL-6 and IL-10, with no effects on other cytokines observed. In addition, Lyn-/- mice with autophagy-deficient myeloid cells showed reduced expression of autoantibodies relevant to systemic lupus erythematosus, including anti-histone and anti-Smith protein. In vitro, loss of autophagy, through pharmacological inhibition or small interfering RNA against Becn1, inhibited IL-10 release by human and mouse myeloid cells. This effect was evident at the level of Il10 messenger RNA expression. Our data highlight potentially important differences in the role of myeloid cell autophagy in acute and chronic inflammation and demonstrate a direct role for autophagy in the production and release of IL-10 by macrophages.

GILZ regulates type I interferon release and sequesters STAT1.

Glucocorticoids remain a mainstay of modern medicine due to their ability to broadly suppress immune activation. However, they cause severe adverse effects that warrant urgent development of a safer alternative. The glucocorticoid-induced leucine zipper (GILZ) gene, TSC22D3, is one of the most highly upregulated genes in response to glucocorticoid treatment, and reduced GILZ mRNA and protein levels are associated with increased severity of inflammation in systemic lupus erythematosus (SLE), Ulcerative Colitis, Psoriasis, and other autoimmune/autoinflammatory diseases. Here, we demonstrate that low GILZ permits expression of a type I interferon (IFN) signature, which is exacerbated in response to TLR7 and TLR9 stimulation. Conversely, overexpression of GILZ prevents IFN-stimulated gene (ISG) up-regulation in response to IFNα. Moreover, GILZ directly binds STAT1 and prevents its nuclear translocation, thereby negatively regulating IFN-induced gene expression and the auto-amplification loop of the IFN response. Thus, GILZ powerfully regulates both the expression and action of type I IFN, suggesting restoration of GILZ as an attractive therapeutic strategy for reducing reliance on glucocorticoids.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

A sprinkle of salt in the pressure cooker of innate immunity and inflammation.

In this issue, Schroder et al. assess the impacts of mechanical strain and salt on macrophage inflammatory responses in vitro. They demonstrate a complex role for the transcription NFAT5 in cytokine release in response to stress, strain and salt in the context of orthodontic treatments.

Necrotic cell death increases the release of macrophage migration inhibitory factor by monocytes/macrophages.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory molecule with both cytokine and noncytokine activity. MIF is constitutively released from multiple cell types via an unconventional secretory pathway that is not well defined. Here, we looked at MIF release from human and mouse monocytes/macrophages in response to different stimuli. While MIF release was not significantly altered in response to lipopolysaccharide or heat-killed Escherichia coli, cytotoxic stimuli strongly promoted release of MIF. MIF release was highly upregulated in cells undergoing necrosis, necroptosis and NLRP3 inflammasome-dependent pyroptosis. Our data suggest that cell death represents a major route for MIF release from myeloid cells. The functional significance of these findings and their potential importance in the context of autoimmune and inflammatory diseases warrant further investigation.

Glucocorticoid-induced leucine zipper modulates macrophage polarization and apoptotic cell clearance.

Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-α, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation.

Treatment of abdominal pseudocysts and associated ventricuoperitoneal shunt failure.

PURPOSE: The purpose of this study was to determine whether drainage and revision are an effective treatment for abdominal pseudocyst associated ventriculoperitoneal (VP) shunt failure by estimating the total rate of secondary shunt failure. METHODS: We performed a retrospective review of children with hydrocephalus diagnosed with and treated for an abdominal pseudocyst at the Children's Hospital, London Health Sciences Centre (LHSC) between January 1, 2000 and May 31, 2016 (ethics approval # 108136). Patients with a VP shunt were included if (i) the development of an abdominal pseudocyst at age 2 to 18 years was identified, (ii) treatment of the pseudocyst by either interventional radiology (IR) or surgical drainage, and (iii) revision of the VP shunt. Demographic data and details of pseudocyst formation/ treatment as well as subsequent failures were identified. RESULTS: Twelve patients who had a VP shunt developed abdominal pseudocyst and met inclusion criteria. A 91% shunt failure rate after drainage and shunt revision was identified. Three patients had the pseudocyst drained in interventional radiology and then externalized due to shunt infection. Nine patients were treated by surgical revision. Ten patients experienced recurrent shunt failure following initial drainage of the pseudocyst: pseudocyst reoccurrence (n = 3), distal obstruction from adhesions (n = 1), and uncleared infection (n = 6). CONCLUSION: The results suggest that pseudocyst drainage and shunt revision is ineffective in providing long-term resolution of shunt problems.

Glucocorticoid-induced leucine zipper (GILZ) inhibits B cell activation in systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus (SLE) is a serious multisystem autoimmune disease, mediated by disrupted B cell quiescence and typically treated with glucocorticoids. We studied whether B cells in SLE are regulated by the glucocorticoid-induced leucine zipper (GILZ) protein, an endogenous mediator of anti-inflammatory effects of glucocorticoids. METHODS: We conducted a study of GILZ expression in blood mononuclear cells of patients with SLE, performed in vitro analyses of GILZ function in mouse and human B cells, assessed the contributions of GILZ to autoimmunity in mice, and used the nitrophenol coupled to keyhole limpet haemocyanin model of immunisation in mice. RESULTS: Reduced B cell GILZ was observed in patients with SLE and lupus-prone mice, and impaired induction of GILZ in patients with SLE receiving glucocorticoids was associated with increased disease activity. GILZ was downregulated in naïve B cells upon stimulation in vitro and in germinal centre B cells, which contained less enrichment of H3K4me3 at the GILZ promoter compared with naïve and memory B cells. Mice lacking GILZ spontaneously developed lupus-like autoimmunity, and GILZ deficiency resulted in excessive B cell responses to T-dependent stimulation. Accordingly, loss of GILZ in naïve B cells allowed upregulation of multiple genes that promote the germinal centre B cell phenotype, including lupus susceptibility genes and genes involved in cell survival and proliferation. Finally, treatment of human B cells with a cell-permeable GILZ fusion protein potently suppressed their responsiveness to T-dependent stimuli. CONCLUSIONS: Our findings demonstrated that GILZ is a non-redundant regulator of B cell activity, with important potential clinical implications in SLE.

GILZ regulates Th17 responses and restrains IL-17-mediated skin inflammation.

Patients with inflammatory autoimmune diseases are routinely treated with synthetic glucocorticoids to suppress immunopathology. A crucial outcome of glucocorticoid exposure is induction of glucocorticoid-induced leucine zipper (GILZ), a protein with multiple functions that include inhibition of key immune cell signalling pathways. Here we report that GILZ maintains a threshold for activation of Th17 responses and IL-17-dependent pathology. GILZ expression was deficient in lesional skin of psoriasis patients and was negatively correlated with the pro-inflammatory cytokines IL-23, IL-17A and IL-22, and with STAT3 expression. Deficiency of GILZ in mice resulted in excessive inflammation and pro-inflammatory cytokine expression in the imiquimod model of psoriasis, and dendritic cells lacking GILZ produced greater IL-1, IL-23 and IL-6 in response to imiquimod stimulation in vitro. These cytokines stimulate Th17 cell differentiation, and we found unchallenged GILZ-deficient mice to have spontaneous production of IL-17A and IL-22 in vivo. We also identified a T cell-intrinsic role for GILZ in limiting Th17 cell formation in vitro in response to Th17-promoting cytokines IL-1ß and IL-23. Addition of IL-6 under these conditions suppressed GILZ, allowing T cell proliferation and expression of Th17 genes, whereas exogenous delivery of GILZ using a cell-permeable fusion protein restored regulation of Th17 cell proliferation. Thus, GILZ has a non-redundant function to constrain pathogenic Th17 responses, with clinical implications for psoriasis.

Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner.

The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.

GILZ: a new link between the hypothalamic pituitary adrenal axis and rheumatoid arthritis?

The hypothalamic-pituitary-adrenal (HPA) axis is an important regulator of the stress response. In healthy individuals, the HPA axis maintains an equilibrium, ensuring that endogenous glucocorticoid (GC) levels remain within the normal range. However, hypofunction of the HPA axis may have a role in the development of inflammatory diseases, such as rheumatoid arthritis (RA). Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein, the expression of which is upregulated by GC. Although GILZ mediates the anti-inflammatory effects of GC, it may not be associated with the adverse effects that are frequently caused by exogenous GC administration. This has raised interest in GILZ potentiation as a therapeutic approach in diseases such as RA, which may mimic the anti-inflammatory effects of GC without causing harmful side effects. This review will outline the involvement of the HPA axis in RA, as a prelude to highlighting emerging evidence regarding the role of GILZ in inflammation control and RA.

Autophagy regulates IL-23 secretion and innate T cell responses through effects on IL-1 secretion.

Autophagy controls IL-1ß secretion by regulating inflammasome activation and by targeting pro-IL-1ß for degradation. In this article, we show that inhibition of autophagy, either with the PI3K inhibitors 3-methyladenine, wortmannin, and LY294002 or with small interfering RNA against autophagy proteins augmented the secretion of IL-23 by human and mouse macrophages and dendritic cells in response to specific TLR agonists. This process occurred at the transcriptional level and was dependent on reactive oxygen species and IL-1R signaling; it was abrogated with an IL-1R antagonist or with IL-1-neutralizing Abs, whereas treatment with either rIL-1α or IL-1ß induced IL-23 secretion. Dendritic cells treated with LPS and 3-methyladenine secreted enhanced levels of both IL-1ß and IL-23, and supernatants from these cells stimulated the innate secretion of IL-17, IFN-γ, and IL-22 by γδ T cells. These data demonstrate that autophagy has a potentially pivotal role to play in the induction and regulation of inflammatory responses by innate immune cells, largely driven by IL-1 and its consequential effects on IL-23 secretion.

Targeting Interferon Signalling in Systemic Lupus Erythematosus: Lessons Learned.

The development of new medicines for systemic lupus erythematosus (SLE) has not addressed unmet clinical need, with only three drugs receiving regulatory approval for SLE in the last 60 years, one of which was specifically licensed for lupus nephritis. In the last 20 years it has become clear that activation of type 1 interferons (IFN) is reproducibly detected in the majority of SLE patients, and the actions of IFN in the immune system and on target tissues is consistent with a pathogenic role in SLE. These findings led to considerable drug discovery activity, first with agents directly targeting IFN family cytokines, with results that were encouraging but underwhelming. In contrast, targeting the type I IFN receptor with the monoclonal antibody anifrolumab, thereby blocking all IFN family members, was effective in a phase II clinical trial. This led to a pair of phase III trials, one of which was negative and the other positive, reflecting the difficulty of obtaining outcomes from trials in this complex disease. Nonetheless, the balance of evidence resulted in approval of anifrolumab in multiple jurisdictions from 2021 onwards. Multiple approaches to targeting the type 1 IFN pathway have subsequently had positive phase II clinical trials, including antibodies targeting cells that produce IFN, and small molecules targeting the receptor kinase TYK2, required for IFN signalling. Despite multiple hurdles, it is clear that IFN targeting in SLE is here to stay. The story of IFN-targeting therapy in SLE has lessons for drug development overall in this disease.

Autophagy and inflammatory diseases.

Autophagy is a cellular mechanism for the sequestration and degradation of intracellular pathogens and compromised organelles, particularly damaged mitochondria. Autophagy also clears other cellular components, such as inflammasomes and cytokines, thus providing an important means of regulating inflammation. Defects in autophagy have been found by genetic association studies to confer susceptibility to several autoimmune and inflammatory disorders, particularly inflammatory bowel disease. Thus, the manipulation of autophagy in disease situations is of growing interest for therapeutic targeting; however, the involvement of autophagy in cellular homoeostasis, in normal immune function and in inflammation is manifold. An appreciation of the intricacies of the contributions of this process to inflammation, and how these are altered by various immune and environmental stimuli, is essential for the understanding and interpretation of studies of inflammation and the design of therapeutics exploiting the manipulation of autophagy. This review focuses on the known roles of autophagy in the induction and maintenance of inflammation and on its role in the aetiology and regulation of inflammatory and autoimmune disorders.

SOCS3 deletion in B cells alters cytokine responses and germinal center output.

B cell behavior is fine-tuned by internal regulatory mechanisms and external cues such as cytokines and chemokines. Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of STAT3-dependent cytokine responses in many cell types and has been reported to inhibit CXCL12-induced retention of immature B cells in the bone marrow. Using mice with SOCS3 exclusively deleted in the B cell lineage (Socs3(Δ/Δ)mb1cre(+)), we analyzed the role of SOCS3 in the response of these cells to CXCL12 and the STAT3-inducing cytokines IL-6 and IL-21. Our findings refute a B cell-intrinsic role for SOCS3 in B cell development, because SOCS3 deletion in the B lineage did not affect B cell populations in naive mice. SOCS3 was strongly induced in B cells stimulated with IL-21 and in plasma cells exposed to IL-6. Its deletion permitted excessive and prolonged STAT3 signaling following IL-6 stimulation of plasma cells and, in a T cell-dependent immunization model, reduced the number of germinal center B cells formed and altered the production of Ag-specific IgM and IgE. These data demonstrate a novel regulatory signal transduction circuit in plasma cells, providing, to our knowledge, the first evidence of how these long-lived, sessile cells respond to the external signals that mediate their longevity.